Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM alphaW62A,betaW120A (DM(W62A/W120A)) double mutant was expressed in HLA-DR(+) HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.     

Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in biological fluids

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.     

Prenatal detection of a fetus hemizygous for the fragile X-chromosome

Summary A male fetus of a pregnancy known to be at risk for X-linked mental retardation with fragile site Xq27 was found to be affected by demonstrating the marker X-chromosome in five of 180 (2.8%) of metaphases derived from amniocytes cultured in medium 199. The results were confirmed in fetal lymphocytes (25 of 86 metaphases, i.e. 29%), and fetal fibroblasts (five of 100 metaphases when cultured in medium 199, and 14 of 100 after exposure to methotrexate for 43 h).This work was supported in part by a research grant from the Deutsche Forschungsgemeinschaft     

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.